Combination therapy for MDS

ABSTRACT

Disclosed are compositions and methods for the treatment of disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The disclosed methods include administering to an individual in need of such treatment a composition that may include an IRAK1/4 inhibitor. In other aspects, the method may include administration of a BLC2 inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application Ser. No.61/826,211, filed May 22, 2013, incorporated herein by reference in itsentirety.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under Grant Nos.RO1HL111103 and P30DK090971. The government has certain rights in thisinvention.

BACKGROUND

Myelodysplastic Syndromes (MDS) are malignant, potentially fatal blooddiseases that arise from a defective hematopoietic stem/progenitor cell.MDS are heterogeneous diseases with few treatment options. One of thekey challenges facing MDS treatment is the lack of effective medicinescapable of providing a durable response.

MDS are hematologic malignancies defined by blood cytopenias due toineffective hematopoiesis, and a predisposition to acute myeloidleukemia (AML) (Corey et al., 2007; Nimer, 2008). MDS is most prominentin individuals over 60 years of age, and as a result of longer lifeexpectancies, the incidence of MDS has escalated in recent years(Sekeres, 2010b). MDS is fatal in majority of patients as a result ofmarrow failure, immune dysfunction, and/or transformation to overtleukemia. Current treatment options for MDS include allogeneic HSCtransplantation, demethylating agents, and immunomodulatory therapies(Ebert, 2010). At present, the only curative treatment for MDS is(hemopoeitic stem cell) HSC transplantation, an option unavailable tomany of the older patients. Instead, these patients receive supportivecare and transfusions to ameliorate their disease complications.Unfortunately, even with this treatment, the MDS clones persist in themarrow and the disease invariably advances (Tehranchi et al., 2010). Foradvanced disease or high-risk MDS, patients may also receiveimmunosuppressive therapy, epigenetic modifying drugs, and/orchemotherapy (Greenberg, 2010). Despite recent progress, most MDSpatients exhibit treatment-related toxicities or relapse (Sekeres,2010a). Overall the efficacy of these treatments is variable, andgenerally life expectancies are only slightly improved as compared tosupportive care.

Approximately 30% of MDS patients also develop aggressive Acute Myeloidleukemia (AML) due to acquisition of additional mutations in thedefective hematopoietic stem/progenitor cell (HSPC) (Greenberg et al.,1997). AML is a cancer of the myeloid line of blood cells, characterizedby the rapid growth of abnormal white blood cells that accumulate in thebone marrow and interfere with the production of normal blood cells. AMLis the most common acute leukemia affecting adults, and its incidenceincreases with age. Although AML is a relatively rare disease,accounting for approximately 1.2% of cancer deaths in the United States,its incidence is expected to increase as the population ages. Severalrisk factors and chromosomal abnormalities have been identified, but thespecific cause is not clear. As an acute leukemia, AML progressesrapidly and is typically fatal within weeks or months if left untreated.The prognosis for AML that arises from MDS has a worse as compared toother types of AML.

Consequently, there is an urgent need to develop targeted therapiescapable of eliminating the MDS-initiating clones, and for treatments andmethod of treating MDS and AML. Identification of molecular targets isessential to improve outcome and eliminate the MDS-causing clones and/orAML. Herein, therapeutic targets and agents for treating MDS and/or AMLare described.

BRIEF SUMMARY

Disclosed are compositions and methods for the treatment of disorderssuch as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).The disclosed methods include administering to an individual in need ofsuch treatment an IRAK1 inhibitor. In other aspects, the treatment mayinclude administration of a BLC2 inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1G. IRAK1 is overexpressed and overactivated in MDS. (FIG. 1A)IRAK1 expression is obtained from two gene expression studies on CD34⁺cells isolated from control and MDS marrows (Hofmann et al., 2002;Pellagatti et al., 2010): Pellagatti (p=0.036), Hoffmann (p=0.05) (FIG.1B) Mononuclear marrow cells from 3 control (MNC) and 5 MDS patientswere examined for total and phosphorylated (p)-T209 IRAK1 byimmunoblotting. (FIG. 1C) MNC from 2 controls, 3 AML patients, and 1 MDSpatient were examined for total and pIRAK1T209 IRAK1 by immunoblotting.(FIG. 1D) MNC and the indicated MDS/AML cell lines were examined fortotal, pIRAK1T209, and pIRAK1S376 by immunoblotting. pIRAK1 and IRAK1were run on parallel gels (left panel). Densitometric values for pIRAK1protein relative to GAPDH are summarized in the right panel. (FIG. 1E)Normal CD34+ cells, MDSL, and MDS patient marrow cells (MDS-02; same asin [B]) were examined for total and pIRAK1T209 IRAK1 by immunoblotting.(FIG. 1F-FIG. 1G) THP1 cells were treated with 1 μg/ml LPS (F) or 10ng/ml IL-1β (G) for the indicated time points and analyzed byimmunoblotting for total and pIRAK1T209 (left panels). Densitometricvalues for pIRAK1 protein relative to IRAK1 are summarized in the rightpanel. Error bars represent ±SD. See also FIG. 8 and Table 1.

FIG. 2A-2H. IRAK-Inh suppresses TRAF6 and NF-κB activation in MDS/AML.(FIG. 2A) Schematic of IRAK1/TRAF6/NF-κB signaling. IRAK-Inh (red line)inhibits IRAK1 kinase function and prevents TRAF6-mediated canonicalNF-κB activation. (FIG. 2B) Protein lysates from THP1 cells and (FIG.2C-FIG. 2D) MDS and AML marrow cells treated with IRAK-Inh for 24 hrswere evaluated by immunoblotting for total and pIRAK1T209. Densitometricvalues for pIRAK1 protein relative to GAPDH or IRAK1 are shown below.(FIG. 2E) THP1 cells were treated with 10 μM IRAK-Inh for 24 hrs wereisolated for immunoprecipitation (IP) of IRAK1. Immunoblot for ubiquitin(Ub) and pIRAK1T209 was performed on the IP lysate. Shown is arepresentative image from 3 independent replicates. Densitometric valuesfor Ub-IRAK1 protein relative to GAPDH are shown below. (FIG. 2F) Emptyvector- and TRAF6-transduced THP1 cells were treated with IRAK-Inh (10μM) for 24 hrs and examined by immunoblotting. (FIG. 2G) Nuclear lysatesfrom THP1 and HL60 cell-lines treated with IRAK-Inh (10 μM) for 24 hrswere evaluated for p65 DNA binding activity. Shown is the mean of 3independent replicates. Error bars represent +/−SEM. (FIG. 2H) THP 1cells were treated with 10 μM IRAK-Inh for 24 hrs were isolated for IPof TRAF6. Immunoblot for ubiquitin (Ub) and TRAF6 was performed on theIP lysate. Shown is a representative image from 3 independentreplicates. Densitometric values for Ub-TRAF6 protein relative to GAPDHare shown below.

FIG. 3A-G. IRAK-Inh impairs MDS cell viability and progenitor function.(FIG. 3A) Viable cell growth of the indicated cell lines and normalCD34⁺ cells was assayed by trypan blue exclusion in the presence ofIRAK-Inh (0-10 μM) for up to 6 days. (FIG. 3B) Relationship betweenIRAK-Inh sensitivity (IC50) and pIRAK1 levels (densitometric values ofimmunoblotted pIRAK1) was calculated for the indicated cell lines. (FIG.3C) Annexin V staining of the indicated cells was determined after 48 hrtreatment with IRAK-Inh (10 μM). (FIG. 3D) Cell cycle analysis of MDSLcells treated with 10 μM IRAK-Inh for 6 days was determined by BrdU/7AADincorporation. (FIG. 3E) Colony formation in methylcellulose wasdetermined for the indicated cell lines treated with IRAK-Inh. Totalcolonies were scored after 10 days. (FIG. 3F-FIG. 3G) Marrow cells fromMDS patients were evaluated for growth (trypan blue exclusion, FIG. 3F)and for apoptosis (Annexin V staining, FIG. 3G) in the presence ofIRAK-Inh. (FIG. 3H) Marrow cells from MDS patients were evaluated forcolony formation in methylcellulose containing IRAK-Inh. *, p<0.05; **,p<0.01; ***, p<0.001. Error bars represent ±SD.

FIG. 4A-H. IRAK-Inh suppresses MDS xenografts. (FIG. 4A) Two experimentschemes were used to test the efficacy of IRAK-Inh in an MDS xenograftmodel: (left arm) MDSL cells were pre-treated for 24 hr followed bytransplantation into either NSG or NSGS mice; or (right arm) NSG micewere transplanted with MDSL cells and at day 6 mice were given IRAK-Inh(2.12 mg/kg i.p. 3×/week). (FIG. 4B) Overall survival was determined forNSG (n=4-5/group) and NSGS (n=3-5/group) mice transplanted with MDSLcells treated with IRAK-Inh. (FIG. 4C) Complete blood counts wereperformed at 68 (NSG) and 28 (NSGS) weeks post xenograftment of NSG andNSGS mice. Shown are red blood cells (RBC), hemoglobin (Hb), hematocrit(HCT), and platelets (PLT) for mice receiving control- orIRAK-Inh-treated MDSL cells. (FIG. 4D) Flow cytometric analysisexamining peripheral human graft in representative DMSO and IRAK-Inh NSGmice 28 days post-transplant. (FIG. 4E) Wright-Giemsa marrow cytospinsand blood smears from represented mice transplanted with control orIRAK-Inh-treated MDSL cells. Smears on all mice were performed at timeof death of the control group. MDSL cells are indicated by arrowheads.Blood scale bar, 30 μm; BM scale bar, 5 μm. (FIG. 4F) NSG animals weretransplanted with 1×10⁶ cells/mouse. Six days post transplant, mice wereinjected with IRAK-Inh (2.12 mg/kg, 3×/week). Hematocrit (HCT) andhemoglobin (Hg) counts were performed on the indicated days (>4 mice pergroup). Error bars represent +/−SD. (FIG. 4G) Human MDSL engraftment wasdetermined by flow cytometry by measuring hCD45 in peripheral blood.(FIG. 4H) Summary of MDSL engraftment in mice receiving IRAK-Inh (n=5per group). *, p<0.05; **, p<0.01.

FIG. 5A-I. IRAK1 is essential for MDS progenitor cell survival. (FIG.5A) IRAK1 knockdown was confirmed by qRT-PCR in cells expressing acontrol or shIRAK1-expressing lentiviral vector. (FIG. 5B) Annexin Vstaining by flow cytometry was measured after transduction with ashIRAK1-expressing lentiviral vector. (FIG. 5C-FIG. 5D)The indicatedcell lines and primary MDS cells were transduced with shRNA-expressinglentiviral vectors (GFP+), sorted for GFP, and then plated inmethylcellulose for progenitor colony formation. Colonies were scored10-14 days after plating. (FIG. 5E) THP1 cells were transduced with alentiviral vector containing a doxycycline (DOX)-inducible promoter,which drives the expression of an IRAK1-targeting shRNA Immunoblottingand qRT-PCR confirm knockdown of IRAK1 upon addition of DOX. (FIG.5F-FIG. 5G) MDSL cells transduced with the inducible shIRAK1 weretransplanted into NSG mice (5×10⁶ /mouse). Half the mice receivedDOX-containing chow 7 days post engraftment. Human MDSL engraftment wasdetermined by flow cytometry by measuring hCD45 in peripheral blood(FIG. 5F) and marrow (FIG. 5G). (Day 60: n=7-8; Day 77: n=7-8). (FIG.5H) Spleen size is shown from a representative experiment at time ofdeath. (FIG. 5I) Overall survival was determined for NSG (n=12/group)mice transplanted with MDSL cells transduced with the inducible shIRAK1(with our without DOX-containing chow). *, p<0.05. Error bars represent+/−SD.

FIG. 6A-F. Integrated gene expression profiling of MDS cells followingIRAK1 inhibition or deletion. (FIG. 6A) Microarray analysis wasperformed on MDSL cells transduced with shIRAK1 (or shRNA control) ortreated for 48 hr with IRAK-Inh (or DMSO). Only the top 50differentially expressed genes are shown. GSEA was performed for bothexperiments in order to determine an overlapping gene signature forIRAK1 depletion. (FIG. 6B) Shown are the three overlapping GSEA profiles(from the top 10) generated from shIRAK1 and IRAK-Inh MDSL genesignatures. (FIG. 6C) Shown are enriched GO pathways generated byToppgene for shIRAK1 and IRAK-Inh MDSL cells. P values are shown onlyfor the top GO pathway in each group. (FIG. 6D) BCL2 mRNA expression inMDSL treated with IRAK1 inhibitor (10 μm) or transduced with shIRAK1adapted from the microarray analysis. Error bars represent ±SD. (FIG.6E) MDSL cells were treated with IRAK1 inhibitor (10 μM) or shIRAK1expression induced with DOX for 24 hr Immunoblot analysis was performedto measure BCL2-family protein levels. (FIG. 6F) Densitometric valuesfor BCL2 protein relative to GAPDH are summarized for the indicated celllines treated with 10 μM IRAK-Inh.

FIG. 7A-G. Combined IRAK1 and Bcl-2 inhibition provides a collaborativecytotoxic effect against MDS and AML cells. (FIG. 7A) Viable cell growthof the indicated cell lines was assayed by trypan blue exclusion in thepresence of IRAK-Inh (10 μM), ABT-263 (0.1 μM) or with the combinationof both drugs. (FIG. 7B) Annexin V/7AAD staining by flow cytometry after48 hr treatment with IRAK-Inh (10 μM), ABT-263 (0.1 μM) or thecombination of both drugs. (FIG. 7C) The indicated cell lines wereevaluated for colony formation in methylcellulose in the presence ofIRAK-Inh (10 μM), ABT-263 (0.1 μM) or the combination of both drugs.(FIG. 7D-FIG. 7F) Marrow cells from MDS (MDS-02) and AML patients(AML-01 and AML-02) were plated in methylcellulose containing IRAK-Inh(10 μM), ABT-263 (0.1 μM), or the combination of both drugs. (G) As inFIG. 4A, overall survival was determined for NSGS mice transplanted withMDSL cells treated with IRAK-Inh, ABT-263, or with the combination ofboth drugs for 72 hrs. To the right, p values are shown for the variousexperimental combinations. *, p<0.05; **, p<0.01; ***, p<0.001. Errorbars represent ±SD.

FIG. 8A-G. Deregulation of IRAK1 in MDS by miR-146a. (FIG. 8A) RNAisolated from MDS patient marrow cells (n=20) was evaluated for IRAK1mRNA. Shown is IRAK1 mRNA from MDS patients relative to the medianexpression from control marrow cells (n=10; dashed line). MDS patientswere divided into low (below 1.0) and high (above 1.0) IRAK1 expression.(FIG. 8B) Overall survival was determined for the MDS patients accordingto high and low IRAK1 expression. (FIG. 8C) IRAK1 mRNA (qRT-PCR), IRAK1protein (immunoblot), and miR-146a (qRT-PCR) were determined for theindicated cells and normalized to normal mononuclear cells (MNC). Errorbars represent +1-SD. (FIG. 8D) MDSL cells were transduced with amiR-146a decoy (pGK-GFP) as previously described (Starczynowski et al.,2010). Endogenous miR-146a expression was reduced by −75% (not shown).Expression of IRAK1 and TRAF6 was determined by immunoblotting andcompared to vector-transduced cells. (FIG. 8E-FIG. 8F) Total (FIG. 8E)and phosphorylated (FIG. 8F) IRAK1 was quantitated and compared tomiR-146a expression in the indicated cells. (FIG. 8G) IRAK1 and miR-146aexpression was compared in MDS patient marrow cells (n=20).

FIG. 9A-C. IRAK-Inh selectively targets IRAK1 in MDS/AML cells and itseffects are partially rescued by overexpression of TRAF6. (FIG. 9A)Protein lysates from MDSL cells treated with IRAK-Inh (0.1M) for 24 hrswere evaluated by immunoblotting for kinases that can be targeted by1RAK-Inh in non-hematopoietic cells and at higher concentrations: IRAK1(IC₅₀=0.3 μM); IRAK4 (IC₅₀=0.2 μM); pp60^(Src) (IC₅₀>10 μM); and Lck(IC₅₀>10 μM) (Powers et al., 2006). Densitometric values forphosphorylated relative to unphosphorylated proteins and GAPDH are shownto the right. (FIG. 9B) THP1 cells were transduced with empty vector-,IRAK1-, or TRAF6-containing LeGO-iG2 lentiviral vectors. Immunoblotswere carried out to confirm IRAK1 and TRAF6 overexpression (FIG. 9C) andto examine pIRAK1 T2(19 and GAPDH when treated with IRAK-Inh (μM)Densitometric values for pIRAK1 relative to GAPDH are shown below andnormalized to 0 μM.

FIG. 10A-K. Effects of IRAK-Inh on primary AML and normal CD34* cells.(FIG. 10A) Primary AML cells were cultured in the presence of 10 μMIRAK-Inh for 48 hr and assayed for apoptosis by 7AAD and Annexin Vstaining. (FIG. 10B) Primary AML samples were cultured for the indicatedtime with 10 μM of IRAK-Inh and assayed for growth by trypan blueexclusion. (FIG. 10C) CD34⁺ cord blood cells, plated with either DMSO,IRAK-Inh (10 μM), ABT-263 (0.1 μM), or with the combination of bothdrugs, were evaluated for colony formation in methylcellulose assay(left panel). Colonies were scored as either granulocytic (G),macrocytic (G), mixed granulocytic/macrocytic (GM), erythroid (E), ormixed granulocytic, erythrocytic, macrocytic, megakaryocytic (GEMM)colonies (right panel). (FIG. 10D) Schematic of experimental design:human CD34⁺ cells and a panel of MDS/AML cell lines were treated withIRAK-Inh (10 μM) for 24 hrs and then plated in methylcellulose. (FIG.10E) Colony formation was determined after 10 days. (FIG. 10F) As in(FIG. 10E), primary MDS patient marrow cells were treated with DMSO orIRAK-Inh (10 μM) for 24 hrs and then plated in methylcellulose to assayprogenitor frequency. (FIG. 10G) Representative cytospins of the MDSLcells exposed to either DMSO or IRAK-Inh (10 μM) for 24 hr. Scale bar, 5μM. (FIG. 10H-FIG. 10I) TRAF6-expressing cells were treated withIRAK-Inh and assessed for cell viability (H; trypan blue exclusion) andcolony formation in methylcellulose (FIG. 10I). (FIG. 10J-FIG. 10K)Colony formation in methylcellulose was determined for 2 AML samplestreated with IRAK-Inh. Total colonies were scored after 10 days. *,p<0.05. Error bars represent ±SD.

FIG. 11A-D. Characterization of MDSL xenograft model. (FIG. 11A) MDSLcells (1×10⁶) were transplanted into NSG mice. At time of disease, bonemarrow (BM), spleen (SP), and blood (PB) were collected and analyzed forengraftment. In addition, BM, SP, and PB were stained withWright-Giemsa. Scale bar, 5 pm. (FIG. 11B) Blood counts performed attime of death for NSGS mice (n=3) indicate pancytopenia. As a control,sublethaly (250Gy) irradiated NSGS mice were transplanted with normalumbilical cord blood (UCB) CD34⁺ cells and blood counts measured at 10weeks post-transplant (n=8). (FIG. 11C) Bone marrow cellularity wasdetermined for healthy NSG mice (HC) (n=6), irradiated mice transplantedwith normal CD34′ cells (n=5), and irradiated mice transplanted withMDSL cells (n=5). Cellularity for mice transplanted with MDSL cells wasdetermined when mice became moribund. (FIG. 11D) Marrow engraftment overtime was determined at the indicated times by performing a femoralaspirate and calculated by quantifying human CD45⁺CD33⁺ cells.

FIG. 12A-J. Lentiviral-mediated knockdown of IRAK1 mRNA and protein.(FIG. 12A) MDSL and CD34⁺ human cord blood cells were transduced withpLKO.1 lentiviral vectors containing 2 independent shRNAs targetingIRAK1. IRAK1 mRNA levels were quantified by qRT-PCR at day 4post-transduction. (FIG. 12B) Immunoblots were performed to quantifyIRAK1 protein expression levels following lentiviral transduction ofTHP1 cells with either empty vector or two independent shRNAs targetingIRAK1. (FIG. 12C) Colony forming assays were carried out on MDSL cellstransduced with pLKO.1 empty vector, or IRAK1-targeting shRNA containingvectors. (FIG. 12D) Domain architecture of doxycycline (DOX)-inducibleTRIPZ shIRAK1 lentiviral construct. TRE, Tet-inducible promoter; RFP,red fluorescent protein; UBC, promoter. The shIRAK1 hairpin targets the3′-UTR of IRAK1. (FIG. 12E) Viable cell growth of the TF-1 cell linetransduced with TRIPZ shIRAK1, in the presence of absence of DOX. (FIG.12F) Apoptosis was evaluated by flow cytometry of TF-1 cells transducedwith TRIPZ shIRAK1 48 hrs after addition of DOX. (FIG. 12G-FIG. 12H) TF1expressing the TRIPZ shIRAK1 were transduced with vector (pLeGO-iG2) or1RAK1 cDNA that is resistant to the shIRAK1 hairpin (lacks the 3′UTRhairpin binding site). Following transduction and sorting (GFP+), cellswere treated with DOX or vehicle control for 5 days and analyzed forIRAK1 expression by immunoblotting (FIG. 12G) and for Annexin V staining(FIG. 12H). The IRAK1 immunoblot is shown after a short and longexposure. (FIG. 12I) Flow cytometric analysis of bone marrow (BM),spleen (SP) and peripheral blood (PB) of moribund NSG animals injectedwith TRIPZ shIRAK1-transduced MDSL. Flow plots were initially gated onviable, and then hCD45+ cells. Increase in RFP indicates expression ofshIRAK1 in vivo. (FIG. 12J) Parental MDSL cells (1×10⁶) weretransplanted into irradiated NSG mice and administered doxycycline(DOX). Addition of DOX did not alter the disease in mice receivingparental MDSL cells. *, p<0.05. Error bars represent ±SD.

FIG. 13A-C. IRAK-Inh and ABT-263 collaborate to induce cytotoxicity.(FIG. 13A) MDSL cells were treated with increasing concentrations ofABT-263 (0, 0.01, 0.1, and 1.0 μM) and IRAK-Inh (0, 0.1, 1.0, 5.0, and10 μM) alone or in combination for 72 hrs. Surviving fraction of MDSLcells was determined by Annexin V/P1 staining and the live fractionvalues are shown inside the box. (FIG. 13B) Cells were treated with 0.1μM ABT-263 and increasing concentration of IRAK-Inh. Note thecooperative cytotoxic effect of 0.1 μM ABT-263 with increasingconcentrations of IRAK-lnh. (FIG. 13C) Cells were treated with 10 μMIRAK-Inh and increasing concentration of ABT-263. Note the cooperativecytotoxic effect of 10 μM IRAK-lnh and 0.1 μM ABT-263. Dotted linerepresents the dose at which 50% of cells are alive for ABT-263 and incombination with IRAK-Inh.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a method” includesa plurality of such methods and reference to “a dose” includes referenceto one or more doses and equivalents thereof known to those skilled inthe art, and so forth.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviations,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, or up to 10%, or up to 5%, or up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, preferablywithin 5-fold, and more preferably within 2-fold, of a value. Whereparticular values are described in the application and claims, unlessotherwise stated the term “about” meaning within an acceptable errorrange for the particular value should be assumed.

“Dosage unit form” as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated, each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe preferred embodiments are dictated by and directly dependent on theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and the limitations inherent in theart of compounding such an active compound for the treatment ofindividuals.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably to refer to an animal that is the object of treatment,observation and/or experiment. “Animal” includes vertebrates andinvertebrates, such as fish, shellfish, reptiles, birds, and, inparticular, mammals. “Mammal” includes, without limitation, mice, rats,rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates,such as monkeys, chimpanzees, and apes, and, in particular, humans.

As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Pharmaceutically acceptable carriers include a wide range of knowndiluents (i.e., solvents), fillers, extending agents, binders,suspending agents, disintegrates, surfactants, lubricants, excipients,wetting agents and the like commonly used in this field. These carriersmay be used singly or in combination according to the form of thepharmaceutical preparation, and may further encompass “pharmaceuticallyacceptable excipients” as defined herein.

As used herein, “pharmaceutically acceptable excipient” means any othercomponent added to a pharmaceutical formulation other than the activeingredient and which is capable of bulking-up formulations that containpotent active ingredients (thus often referred to as “bulking agents,”“fillers,” or “diluents”) to allow convenient and accurate dispensationof a drug substance when producing a dosage form. Excipients may beadded to facilitate manufacture, enhance stability, control release,enhance product characteristics, enhance bioavailability drug absorptionor solubility, or other pharmacokinetic considerations, enhance patientacceptability, etc. Pharmaceutical excipients include, for example,carriers, fillers, binders, disintegrants, lubricants, glidants, colors,preservatives, suspending agents, dispersing agents, film formers,buffer agents, pH adjusters, preservatives etc. The selection ofappropriate excipients also depends upon the route of administration andthe dosage form, as well as the active ingredient and other factors, andwill be readily understood by one of ordinary skill in the art.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, e.g.,healing of chronic conditions or in an increase in rate of healing ofsuch conditions, or in a reduction in aberrant conditions. This includesboth therapeutic and prophylactic treatments. Accordingly, the compoundscan be used at very early stages of a disease, or before early onset, orafter significant progression. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the therapeutic effect,whether administered in combination, serially or simultaneously.

Targeted therapies have been effective in other myeloid diseases(O'Dwyer et al., 2003), and may also improve the clinical outcome in MDSby suppressing the malignant clone. Recent sequencing and gene profilingefforts have revealed insight into the underlying molecular and cellulardefects in MDS-initiating cells. Despite this progress, one of the keychallenges still facing MDS treatment is that molecular-targetedtherapies do not exist and AML-like therapies have been disappointing.

MDS are genetically defined by somatic mutations and chromosomalabnormalities not only affecting epigenetic plasticity, ribosomefunction, spliceosome machinery, or activation of oncogenes but alsoimmune dysfunction. Human miR-146a resides on chromosome 5q33.3, and itsdeletion occurs in 80% of all del(5q) MDS and AML (Gondek et al., 2008).Low expression of miR-146a, also occurs in >25% of all MDS and in >10%of AML patients (Sokol et al., 2011; Starczynowski et al., 2010;Starczynowski et al., 2011b), and is part of an MDS diagnostic miRNAsignature (Sokol et al., 2011). Knockout of miR-146a results in an earlyonset of myeloid expansion in the marrow, and progression to moreaggressive diseases such as lymphomas, marrow failure, and myeloidleukemia (Boldin et al., 2011; Zhao et al., 2011).

TRAF6 and IRAK1 are two immune-related targets of miR-146a(Starczynowski et al., 2010; Starczynowski et al., 2011a; Taganov etal., 2006), and as expected, miR-146a knockout mice have a dramaticincrease in TRAF6 and IRAK1 protein within the hematopoietic compartment(Boldin et al., 2011; Zhao et al., 2011). TRAF6, a lysine (K)-63 E3ubiquitin ligase, and IRAK1, a serine/threonine kinase, are interactingproteins and mediators downstream of Toll-like (TLR) and Interleukin-1(IL1R) receptors. Activation of TLR or IL1R recruits a series of adaptorproteins resulting in phosphorylation of IRAK1 on Thr209. PhosphorylatedIRAK1 binds to and activates TRAF6 resulting in NF-κB activation.Increasing clinical and biological data indicate that innate immunesignaling is an important determinant of MDS pathophysiology (Bar etal., 2008; Chen et al., 2004; Hofmann et al., 2002; Vasikova et al.,2010).

Here, Applicant has identified that Interleukin Receptor AssociatedKinase-1 (IRAK1) is overexpressed and activated in MDS. Genetic orpharmacological inhibition of IRAK1 induces apoptosis and cell cyclearrest of MDS cells, and prolongs survival outcome in a human MDSxenograft model. In an attempt to understand the mechanism ofIRAK1/4-Inh function and potential resistance, Applicant identified acollaborative cytotoxic effect of combined IRAK1 and BCL2 inhibition onMDS cells. Applicant's findings suggest that targeting IRAK1 may be aneffective therapeutic strategy in MDS.

In particular, Applicant identified that IRAK1, an immune modulatingkinase, is overexpressed and hyperactivated in MDS. MDS clones treatedwith a small-molecule IRAK1 inhibitor (IRAK1/4-Inh) exhibited impairedexpansion and increased apoptosis, which coincided with TRAF6/NF-κBinhibition. Suppression of IRAK1, either by RNAi or with IRAK1/4-Inh, isdetrimental to MDS cells while sparing normal CD34+ cells. Based on anintegrative gene expression analysis, we combined IRAK1 and BCL2inhibitors and found that co-treatment more effectively eliminated MDSclones. In summary, these findings implicate IRAK1 as a drugable targetin MDS.

Disclosed herein are methods for treating myelodysplastic syndrome (MDS)in an individual. The method may comprise the step of administering tothe individual a composition that may comprise an IRAK1 inhibitor.

In one aspect, the IRAK1/4 inhibitor may be selected fromN-acyl-2-aminobenzimidazoles, imidazo[1,2-a]pyridino-pyrimidine,imidazo[1,2-a]pyridino-pyridine, benzimidazolo-pyridine,N-(2-morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole, (IRAK1/4),or combinations thereof.

In one aspect, the IRAK1/4 inhibitor may comprise an RNAi sufficient toinhibit IRAK1 expression.

In one aspect, the method may comprise the step of administering to theindividual an apoptotic modulator.

In one aspect, the method may comprise the step of administering to saidindividual an apoptotic modulator. The apoptotic modulator comprises maycomprise a BCL2 inhibitor.

In one aspect, the method may comprise the step of administering to theindividual an apoptotic modulator, wherein the apoptotic modulator maycomprise a BCL2 inhibitor selected from ABT-263 (Navitoclax), ABT-737,ABT-199, GDC-0199, GX15-070 (Obatoclax), and combinations thereof, allavailable from Abbott Laboratories.

In one aspect, the myelodysplastic syndrome may be selected from FanconiAnemia, refractory anemia, refractory neutropenia, refractorythrombocytopenia, refractory anemia with ring sideroblasts (RARS),refractory cytopenia with multilineage dysplasia (RCMD), refractoryanemia with excess blasts I and II (RAEB), 5q-syndrome, myelodysplasiaunclassifiable, refractory cytopenia of childhood, or a combinationthereof.

In one aspect, the administering step may be selected from orally,rectally, nasally, topically, parenterally, subcutaneously,intramuscularly, intravenously, transdermally, or a combination thereof.

In one aspect, the administration may decreases the incidence of marrowfailure, immune dysfunction, transformation to overt leukemia, orcombinations thereof in said individual, as compared to an individualnot receiving said composition.

In one aspect, the method may decrease a marker of viability of MDScells. In one aspect, the method may decrease a marker of viability ofMDS and/or AML cells. The marker may be selected from survival overtime, proliferation, growth, migration, formation of colonies, chromaticassembly, DNA binding, RNA metabolism, cell migration, cell adhesion,inflammation, or a combination thereof.

In one aspect, a method of treating myelodysplastic syndrome or acutemyeloid leukemia in an individual is disclosed. In this aspect, themethod may comprise the step of administering to the individual

a) an IRAK1/4 inhibitor; and

b) an agent selected from an apoptotic agent, an immune modulatingagent, an epigenetic modifying agent, and combinations thereof.

In one aspect, the IRAK1/4 inhibitor may be selected fromN-acyl-2-aminobenzimidazoles, imidazo[1,2-a]pyridino-pyrimidine,imidazo[1,2-a]pyridino-pyridine, benzimidazolo- pyridine,N-(2-morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole, (IRAK1/4),or combinations thereof. In one aspect, the IRAK1 inhibitor may comprisean RNAi sufficient to inhibit IRAK1 expression.

In one aspect, the administration may comprise administration of anapoptotic modulator. The apoptotic modulator may comprise a BLC2inhibitor. In certain aspects, the apoptotic modulator may be selectedfrom ABT-263 (Navitoclax), ABT-737, ABT-199, GDC-0199, GX15-070,(Obatoclax), and combinations thereof.

In one aspect, the administration step may comprise administration of animmune modulator. The immune modulator may comprise, for example,Lenalidomide (Revlamid; Celgene Corporation).

In one aspect, the administration step may comprise administration of anepigenetic modulator. The epigenetic modulator may comprise, forexample, a hypomethylating agent such as azacitidine, decitabine, or acombination thereof.

The myelodysplastic syndrome may be selected from Fanconi Anemia,refractory anemia, refractory neutropenia, refractory thrombocytopenia,refractory anemia with ring sideroblasts (RARS), refractory cytopeniawith multilineage dysplasia (RCMD), refractory anemia with excess blastsI and II (RAEB), 5q-syndrome, myelodysplasia unclassifiable, refractorycytopenia of childhood, or a combination thereof.

The disclosed methods may decreases the incidence of marrow failure,immune dysfunction, transformation to overt leukemia, or combinationsthereof in an individual, as compared to an individual not receiving thedisclosed composition.

In one aspect, a method of identifying a compound useful for treatmentof a myelodysplastic syndrome is disclosed. In this aspect, the methodmay comprise the steps of

a) contacting a MDSL cell with a compound of interest;

b) transplanting the MDSL cell into a host animal;

c) assaying a marker of MDS in the host animal;

wherein a decrease in an incidence of the marker of MDS indicates thatsaid compound of interest is a potential therapeutic agent.

In another aspect, a method of identifying a compound useful fortreatment of a myelodysplastic syndrome is disclosed. In this aspect,the method may comprise the steps of

a) contacting a host animal comprising MDSL cells with a compound ofinterest;

b) assaying a marker of MDS in said host animal;

wherein a decrease in an incidence of the marker of MDS indicates thatthe compound of interest is a potential therapeutic agent.

In certain aspects, the marker may comprise, for example, anemia,thrombocytopenia, hypocellular marrow, extramedullary hematopoiesis, ora combination thereof. In other aspects, the marker may compriseviability or overall health of the host animal

The host animal may be, for example, a mammal, preferably a rodent, morepreferably a mouse. In certain aspects, the host animal may be an NSG orNSGS immunodeficient mouse.

Compositions

In one aspect, a composition is disclosed. The composition may comprisean IRAK1/4 inhibitor and a pharmaceutically acceptable excipient or apharmaceutically acceptable carrier. In other aspects, the compositionmay further comprise an agent selected from an apoptotic agent, animmune modulating agent, an epigenetic modifying agent, and combinationsthereof.

Compounds, or mixtures of compounds described herein, can be formulatedinto pharmaceutical composition comprising a pharmaceutically acceptablecarrier and other excipients as apparent to the skilled worker. Suchcomposition can additionally contain effective amounts of othercompounds, especially for the treatment of conditions, diseases, and/ordisorders described herein.

Some embodiments comprise the administration of a pharmaceuticallyeffective quantity of active agent or its pharmaceutically acceptablesalts or esters, active agent analogs or their pharmaceuticallyacceptable salts or esters, or a combination thereof.

The compositions and preparations may contain at least 0.1% of activeagent. The percentage of the compositions and preparations can, ofcourse, be varied, and can contain between about 2% and 60% of theweight of the amount administered. The percentage of the compositionsand preparations may contain between about 2, 5, 10, or 15% and 30, 35,40, 45, 50, 55, or 60% of the weight of the amount administered. Theamount of active compounds in such pharmaceutically useful compositionsand preparations is such that a suitable dosage will be obtained.

The disclosed active agents may form salts. Reference to a compound ofthe active agent herein is understood to include reference to saltsthereof, unless otherwise indicated. The term “salt(s)”, as employedherein, denotes acidic and/or basic salts formed with inorganic and/ororganic acids and bases. In addition, when an active agent contains botha basic moiety, such as, but not limited to an amine or a pyridine orimidazole ring, and an acidic moiety, such as, but not limited to acarboxylic acid, zwitterions (“inner salts”) can be formed and areincluded within the term “salt(s)” as used herein. Pharmaceuticallyacceptable (e.g., non-toxic, physiologically acceptable) salts arepreferred, although other salts are also useful, e.g., in isolation orpurification steps, which can be employed during preparation. Salts ofthe compounds of the active agent can be formed, for example, byreacting a compound of the active agent with an amount of acid or base,such as an equivalent amount, in a medium such as one in which the saltprecipitates or in an aqueous medium followed by lyophilization.

Pharmaceutically acceptable salts include, but are not limited to,pharmaceutically acceptable acid addition salts, pharmaceuticallyacceptable base addition salts, pharmaceutically acceptable metal salts,ammonium and alkylated ammonium salts. Acid addition salts include saltsof inorganic acids as well as organic acids. Representative examples ofsuitable inorganic acids include hydrochloric, hydrobromic, hydroiodic,phosphoric, sulfuric, nitric acids and the like. Representative examplesof suitable organic acids include formic, acetic, trichloroacetic,trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric,glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric,pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric,ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic,citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic,glutamic, benzenesulfonic, p-toluenesulfonic acids, sulphates, nitrates,phosphates, perchlorates, borates, acetates, benzoates,hydroxynaphthoates, glycerophosphates, ketoglutarates and the like.Examples of metal salts include lithium, sodium, potassium, magnesiumsalts and the like. Examples of ammonium and alkylated ammonium saltsinclude ammonium, methylammonium, dimethylammonium, trimethylammonium,ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium,tetramethylammonium salts and the like. Examples of organic basesinclude lysine, arginine, guanidine, diethanolamine, choline and thelike.

The compounds can be formulated in various forms, including solid andliquid forms, such as tablets, gel, syrup, powder, aerosol, etc.

The compositions may contain physiologically acceptable diluents,fillers, lubricants, excipients, solvents, binders, stabilizers, and thelike. Diluents that can be used in the compositions include but are notlimited to dicalcium phosphate, calcium sulphate, lactose, cellulose,kaolin, mannitol, sodium chloride, dry starch, powdered sugar and forprolonged release tablet-hydroxy propyl methyl cellulose (HPMC). Thebinders that can be used in the compositions include but are not limitedto starch, gelatin and fillers such as sucrose, glucose, dextrose andlactose.

Natural and synthetic gums that can be used in the compositions includebut are not limited to sodium alginate, ghatti gum, carboxymethylcellulose, methyl cellulose, polyvinyl pyrrolidone and veegum.Excipients that can be used in the compositions include but are notlimited to microcrystalline cellulose, calcium sulfate, dicalciumphosphate, starch, magnesium stearate, lactose, and sucrose. Stabilizersthat can be used include but are not limited to polysaccharides such asacacia, agar, alginic acid, guar gum and tragacanth, amphotsics such asgelatin and synthetic and semi-synthetic polymers such as carbomerresins, cellulose ethers and carboxymethyl chitin.

Solvents that can be used include but are not limited to Ringerssolution, water, distilled water, dimethyl sulfoxide to 50% in water,propylene glycol (neat or in water), phosphate buffered saline, balancedsalt solution, glycol and other conventional fluids.

The dosages and dosage regimen in which the compounds are administeredwill vary according to the dosage form, mode of administration, thecondition being treated and particulars of the patient being treated.Accordingly, optimal therapeutic concentrations will be best determinedat the time and place through routine experimentation.

The compounds may also be used enterally. Orally, the compounds may beadministered at the rate of 100 μg to 100 mg per day per kg of bodyweight. Orally, the compounds may be suitably administered at the rateof about 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg to about 1,5, 10, 25, 50, 75, 100 mg per day per kg of body weight. The requireddose can be administered in one or more portions. For oraladministration, suitable forms are, for example, tablets, gel, aerosols,pills, dragees, syrups, suspensions, emulsions, solutions, powders andgranules; one method of administration includes using a suitable formcontaining from 1 mg to about 500 mg of active substance. In one aspect,administration may comprise using a suitable form containing from about1, 2, 5, 10, 25, or 50 mg to about 100, 200, 300, 400, 500 mg of activesubstance.

The compounds may also be administered parenterally in the form ofsolutions or suspensions for intravenous or intramuscular perfusions orinjections. In that case, the compounds may be administered at the rateof about 10 μg to 10 mg per day per kg of body weight; one method ofadministration may consist of using solutions or suspensions containingapproximately from 0.01 mg to 1 mg of active substance per ml. Thecompounds may be administered at the rate of about 10, 20, 30, 40, 50,60, 70, 80, 90, or 100 μg to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg per dayper kg of body weight; in one aspect, solutions or suspensionscontaining approximately from 0.01, 0.02, 0.03, 0.04, or 0.5 mg to 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg of active substance perml may be used.

The compounds can be used in a substantially similar manner to otherknown anti-cancer agents for treating (both chemopreventively andtherapeutically) various cancers. For the anti-cancer dose to beadministered, whether a single dose, multiple dose, or a daily dose,will of course vary with the particular compound employed because of thevarying potency of the compound, the chosen route of administration, thesize of the recipient, the type of cancer, and the nature of thepatient's condition. The dosage to be administered is not subject todefinite bounds, but it will usually be an effective amount, or theequivalent on a molar basis of the pharmacologically active free formproduced from a dosage formulation upon the metabolic release of theactive drug to achieve its desired pharmacological and physiologicaleffects. For example, an oncologist skilled in the art of cancertreatment will be able to ascertain, without undue experimentation,appropriate protocols for the effective administration of the compoundsrelated to cancer therapy, such as by referring to the earlier publishedstudies on compounds found to have anti-cancer properties.

The active compounds and/or pharmaceutical compositions of theembodiments disclosed herein can be administered according to variousroutes, such as by injection, for example local or systemicinjection(s). Intratumoral injections may be used for treating existingcancers. Other administration routes can be used as well, such asintramuscular, intravenous, intradermic, subcutaneous, etc. Furthermore,repeated injections can be performed, if needed, although it is believedthat limited injections will be needed in view of the efficacy of thecompounds.

For ex vivo administration, the active agent can be administered by anystandard method that would maintain viability of the cells, such as byadding it to culture medium (appropriate for the target cells) andadding this medium directly to the cells. As is known in the art, anymedium used in this method can be aqueous and non-toxic so as not torender the cells non-viable. In addition, it can contain standardnutrients for maintaining viability of cells, if desired. For in vivoadministration, the complex can be added to, for example, to apharmaceutically acceptable carrier, e.g., saline and buffered saline,and administered by any of several means known in the art. Examples ofadministration include parenteral administration, e.g., by intravenousinjection including regional perfusion through a blood vessel supplyingthe tissues(s) or organ(s) having the target cell(s), or by inhalationof an aerosol, subcutaneous or intramuscular injection, topicaladministration such as to skin wounds and lesions, direct transfectioninto, e.g., bone marrow cells prepared for transplantation andsubsequent transplantation into the subject, and direct transfectioninto an organ that is subsequently transplanted into the subject.Further administration methods include oral administration, particularlywhen the active agent is encapsulated, or rectal administration,particularly when the active agent is in suppository form.

It is contemplated that such target cells can be located within asubject or human patient, in which case a safe and effective amount ofthe active agent, in pharmacologically acceptable form, would beadministered to the patient. Generally speaking, it is contemplated thatuseful pharmaceutical compositions may include the selected activecompound derivative in a convenient amount, e.g., from about 0.001% toabout 10% (w/w) that is diluted in a pharmacologically orphysiologically acceptable carrier, such as, for example, phosphatebuffered saline. The route of administration and ultimate amount ofmaterial that is administered to the subject under such circumstanceswill depend upon the intended application and will be apparent to thoseof skill in the art in light of the examples which follow.

Any composition chosen should be of low or non-toxicity to the cell.Toxicity for any given compound can vary with the concentration ofcompound used. It is also beneficial if the compound chosen ismetabolized or eliminated by the body and if this metabolism orelimination is done in a manner that will not be harmfully toxic.

The compound may be administered such that a therapeutically effectiveconcentration of the compound is in contact with the affected cells ofthe body. The dose administered to a subject, particularly a human, maybe sufficient to effect a therapeutic response in the subject over areasonable period of time. The dose may be determined by the strength ofthe particular compound employed and the condition of the subject, aswell as the body weight of the subject to be treated. The existence,nature, and extent of any adverse side effects that might accompany theadministration of a particular compound also will determine the size ofthe dose and the particular route of administration employed with aparticular patient. In general, the compounds may be therapeuticallyeffective at low doses. The generally useful dose range may be fromabout 0.001 mM, or less, to about 100 mM, or more. The effective doserange may be from about 0.01, 0.05, 0.1, 0.5, 0.6, 0.7, 0.8, or 0.9 mM,to about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM. Accordingly, the compoundsmay be generally administered in low doses.

The pharmaceutical composition may further comprise a pharmaceuticallyacceptable carrier. The resulting preparation may incorporate, ifnecessary, one or more solubilizing agent, buffers, preservatives,colorants, perfumes, flavorings and the like that are widely used in thefield of pharmaceutical preparation.

The proportion of the active ingredient to be contained in the disclosedcompositions may be determined by one of ordinary skill in the art usingart recognized methods.

The disclosed compounds may be formulated into a dosage form selectedfrom the group consisting of tablets, capsules, granules, pills,injections, solutions, emulsions, suspensions, and syrups. The form andadministration route for the pharmaceutical composition are not limitedand can be suitably selected. For example, tablets, capsules, granules,pills, syrups, solutions, emulsions, and suspensions may be administeredorally. Additionally, injections (e.g. subcutaneous, intravenous,intramuscular, and intraperitoneal) may be administered intravenouslyeither singly or in combination with a conventional replenishercontaining glucose, amino acid and/or the like, or may be singlyadministered intramuscularly, intracutaneously, subcutaneously and/orintraperitoneally.

The disclosed compositions may be prepared according to a method knownin the pharmaceutical field of this kind using a pharmaceuticallyacceptable carrier. For example, oral forms such as tablets, capsules,granules, pills and the like are prepared according to known methodsusing excipients such as saccharose, lactose, glucose, starch, mannitoland the like; binders such as syrup, gum arabic, sorbitol, tragacanth,methylcellulose, polyvinylpyrrolidone and the like; disintegrates suchas starch, carboxymethylcellulose or the calcium salt thereof,microcrystalline cellulose, polyethylene glycol and the like; lubricantssuch as talc, magnesium stearate, calcium stearate, silica and the like;and wetting agents such as sodium laurate, glycerol and the like.

Injections, solutions, emulsions, suspensions, syrups and the like maybe prepared according to a known method suitably using solvents fordissolving the active ingredient, such as ethyl alcohol, isopropylalcohol, propylene glycol, 1,3-butylene glycol, polyethylene glycol,sesame oil and the like; surfactants such as sorbitan fatty acid ester,polyoxyethylenesorbitan fatty acid ester, polyoxyethylene fatty acidester, polyoxyethylene of hydrogenated castor oil, lecithin and thelike; suspending agents such as cellulose derivatives includingcarboxymethylcellulose sodium, methylcellulose and the like, naturalgums including tragacanth, gum arabic and the like; and preservativessuch as parahydroxybenzoic acid esters, benzalkonium chloride, sorbicacid salts and the like.

The compounds can be administered orally, topically, parenterally, byinhalation or spray, vaginally, rectally or sublingually in dosage unitformulations. The term “administration by injection” includes but is notlimited to: intravenous, intraarticular, intramuscular, subcutaneous andparenteral injections, as well as use of infusion techniques. Dermaladministration can include topical application or transdermaladministration. One or more compounds can be present in association withone or more non-toxic pharmaceutically acceptable carriers and ifdesired other active ingredients.

Compositions intended for oral use can be prepared according to anysuitable method known to the art for the manufacture of pharmaceuticalcompositions. Such compositions can contain one or more agents selectedfrom the group consisting of diluents, sweetening agents, flavoringagents, coloring agents and preserving agents in order to providepalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients that aresuitable for the manufacture of tablets. These excipients can be, forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, corn starch, or alginic acid; andbinding agents, for example magnesium stearate, stearic acid or talc.The tablets can be uncoated or they can be coated by known techniques todelay disintegration and adsorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monostearate or glyceryl distearatecan be employed. These compounds can also be prepared in solid, rapidlyreleased form.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions containing the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions can alsobe used. Such excipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethylene oxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolsuch as polyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. The aqueoussuspensions can also contain one or more preservatives, for exampleethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents, and one or more sweetening agents, such assucrose or saccharin.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example, sweetening, flavoring and coloringagents, can also be present.

The compounds can also be in the form of non-aqueous liquidformulations, e.g., oily suspensions which can be formulated bysuspending the active ingredients in a vegetable oil, for examplearachis oil, olive oil, sesame oil or peanut oil, or in a mineral oilsuch as liquid paraffin. The oily suspensions can contain a thickeningagent, for example beeswax, hard paraffin or cetyl alcohol. Sweeteningagents such as those set forth above, and flavoring agents can be addedto provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Compounds may also be administrated transdermally using methods known tothose skilled in the art. For example, a solution or suspension of anactive agent in a suitable volatile solvent optionally containingpenetration enhancing agents can be combined with additional additivesknown to those skilled in the art, such as matrix materials andbacteriocides. After sterilization, the resulting mixture can beformulated following known procedures into dosage forms. In addition, ontreatment with emulsifying agents and water, a solution or suspension ofan active agent can be formulated into a lotion or salve.

Suitable solvents for processing transdermal delivery systems are knownto those skilled in the art, and include lower alcohols such as ethanolor isopropyl alcohol, lower ketones such as acetone, lower carboxylicacid esters such as ethyl acetate, polar ethers such as tetrahydrofuran,lower hydrocarbons such as hexane, cyclohexane or benzene, orhalogenated hydrocarbons such as dichloromethane, chloroform,trichlorotrifluoroethane, or trichlorofluoroethane. Suitable solventscan also include mixtures of one or more materials selected from loweralcohols, lower ketones, lower carboxylic acid esters, polar ethers,lower hydrocarbons, halogenated hydrocarbons.

Suitable penetration enhancing materials for transdermal delivery systemare known to those skilled in the art, and include, for example,monohydroxy or polyhydroxy alcohols such as ethanol, propylene glycol orbenzyl alcohol, saturated or unsaturated C8-C18 fatty alcohols such aslauryl alcohol or cetyl alcohol, saturated or unsaturated C8-C18 fattyacids such as stearic acid, saturated or unsaturated fatty esters withup to 24 carbons such as methyl, ethyl, propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tertbutyl or monoglycerin esters of acetic acid,capronic acid, lauric acid, myristinic acid, stearic acid, or palmiticacid, or diesters of saturated or unsaturated dicarboxylic acids with atotal of up to about 24 carbons such as diisopropyl adipate, diisobutyladipate, diisopropyl sebacate, diisopropyl maleate, or diisopropylfumarate. Additional penetration enhancing materials includephosphatidyl derivatives such as lecithin or cephalin, terpenes, amides,ketones, ureas and their derivatives, and ethers such as dimethylisosorbid and diethyleneglycol monoethyl ether. Suitable penetrationenhancing formulations can also include mixtures of one or morematerials selected from monohydroxy or polyhydroxy alcohols, saturatedor unsaturated C8-C18 fatty alcohols, saturated or unsaturated C8-C18fatty acids, saturated or unsaturated fatty esters with up to 24carbons, diesters of saturated or unsaturated discarboxylic acids with atotal of up to 24 carbons, phosphatidyl derivatives, terpenes, amides,ketones, ureas and their derivatives, and ethers.

Suitable binding materials for transdermal delivery systems are known tothose skilled in the art and include polyacrylates, silicones,polyurethanes, block polymers, styrenebutadiene copolymers, and naturaland synthetic rubbers. Cellulose ethers, derivatized polyethylenes, andsilicates can also be used as matrix components. Additional additives,such as viscous resins or oils can be added to increase the viscosity ofthe matrix.

Pharmaceutical compositions may also be in the form of oil-in-wateremulsions. The oil phase can be a vegetable oil, for example olive oilor arachis oil, or a mineral oil, for example, liquid paraffin ormixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example, gum acacia or gum tragacanth,naturally-occurring phosphatides, for example, soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example, sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for example,polyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents. Syrups and elixirs can be formulatedwith sweetening agents, for example glycerol, propylene glycol, sorbitolor sucrose. Such formulations can also contain a demulcent, apreservative and flavoring and coloring agents.

The compounds can also be administered in the form of suppositories forrectal or vaginal administration of the drug. These compositions can beprepared by mixing the drug with a suitable nonirritating excipientwhich is solid at ordinary temperatures but liquid at the rectaltemperature or vaginal temperature and will therefore melt in the rectumor vagina to release the drug. Such materials include cocoa butter andpolyethylene glycols.

It will be appreciated by those skilled in the art that the particularmethod of administration will depend on a variety of factors, all ofwhich are considered routinely when administering therapeutics. It willalso be understood, however, that the specific dose level for any givenpatient will depend upon a variety of factors, including, the activityof the specific compound employed, the age of the patient, the bodyweight of the patient, the general health of the patient, the gender ofthe patient, the diet of the patient, time of administration, route ofadministration, rate of excretion, drug combinations, and the severityof the condition undergoing therapy. It will be further appreciated byone skilled in the art that the optimal course of treatment, i.e., themode of treatment and the daily number of doses of an active agent or apharmaceutically acceptable salt thereof given for a defined number ofdays, can be ascertained by those skilled in the art using conventionaltreatment tests.

EXAMPLES

Materials and Methods

Cell Lines and CD34+ Cells

Acute myeloid leukemic cell lines, HL60, THP1, and TF-1 were purchasedfrom the American Type Culture Collection. MOLM13 were purchased fromAddexBio. The myelodysplastic cell line, MDS-L, was provided by Dr.Kaoru Tohyama (Kawasaki Medical School, Okayama, Japan) (Matsuoka etal., 2010). Cell-lines were cultured in RPMI 1640 medium with 10% FBSand 1% penicillin-streptomycin. Additionally, both the MDSL and TF-1cell lines were cultured in the presence of 10 ng/mL human recombinantIL-3 (Stemcell Technologies). Human CD34+ umbilical cord blood (UCB) andadult marrow-derived mononuclear cells were obtained from theTranslational Research Development Support Laboratory of CincinnatiChildren's Hospital under an approved Institutional Review Boardprotocol. Human CD34+ UCB cells and primary MDS/AML cells weremaintained in StemSpan Serum-Free Expansion Media (StemcellTechnologies) supplemented with 10 ng/mL of recombinant human stem cellfactor (SCF), Flt3 ligand (Flt3L), thrombopoietin (TPO), IL-3, and IL-6(Stemcell Technologies).

Reagents

The IRAK1 inhibitor (IRAK1/4 inhibitor or IRAK-Inh; Amgen Inc.) waspurchased from Sigma-Aldrich (15409). Lipopolysaccharide was purchasedfrom InvivoGen (TLRL-PEKLPS). ABT-263 (Navitoclax) was purchased fromChemietek (CT-A263) (Tse et al., 2008). Recombinant human IL1-β waspurchased from PeproTech (200-01B). LeGO-iG2 lentiviral vectors forexpression of IRAK1 and TRAF6 cDNAs are described previously (Fang etal., 2012).

Survival Analysis and Growth Curves

Annexin V analysis was carried out as previously described (Fang et al.,2012). Cells were stained after either drug treatment or lentiviraltransduction with Annexin V (eBioscience) and propidium iodide(Sigma-Aldrich), or 7AAD (eBioscience) according to the manufacturer'sinstructions. Analysis was performed using BD FACSCalibur or FACSCantoflow cytometer with either CellQuest or Diva software. For in vitrogrowth assays, cell expansion in liquid culture was determined based ontrypan blue exclusion using an automated cell counter (BioRad TC10). Forexperiments beyond 48 hrs, cells were replenished with fresh media anddrug every second day. For primary patient marrow cells, cell weretreated with a single inhibitor dose and counted 24 and 48 hours later.NF-κB DNA-binding assay Nuclear lysates were isolated from treated cellsas previously described (Starczynowski et al., 2011). NF-κB (p65) DNAbinding was measured using an ELISA-based assay according to themanufacturer's recommendations (KHO0371, Invitrogen)

Immunoblotting and Immunoprecipitation

Total protein lysates were obtained from cells by lysing the samples incold RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, mM EDTA, 1% Triton X-100and 0.1% SDS), in the presence of PMSF, sodium orthovanadate, proteaseand phosphatase inhibitors. After being re-suspended in RIPA cells werebriefly sonicated. Protein concentration was evaluated by a BCA assay(Pierce). For immunoprecipitation, TRAF6 or IRAK1 antibodies (2 μg) wereadded to cell lysates (10 mg) for 3 h at 4° C. and captured by theaddition of Protein A/G Plus beads (sc-2003; Santa Cruz) as describedbefore (Starczynowski et al., 2011). The immune complexes were washedwith lysis buffer followed by the addition of SDS sample buffer. Thebound proteins were separated by SDS-polyacrylamide gel electrophoresis,transferred to nitrocellulose membranes, and analyzed by immunoblotting.Western blot analysis was performed with the following antibodies: TRAF6(sc-7221; Santa Cruz), IRAK1 (sc-7883; Santa Cruz), ubiquitin (sc-8017;Santa Cruz), IKKβ (2370; Cell Signaling Technology), GAPDH (5174; CellSignaling Technology), p38 MAPK (9212; Cell Signaling Technology),phopho-p38 MAPK (4631; Cell Signaling Technology), phospho-IKKα/β (2697;Cell Signaling Technology), phospho-IRAK1 (S376) (PAB0497; Abnova),phospho-IRAK1 (T209) (A1074; AssaybioTech), phospho-ERK (4695; CellSignaling Technology), Lck (2752; Cell Signaling Technology),phospho-Lck (Tyr505) (2751; Cell Signaling Technology), IRAK4 (4363;Cell Signaling Technology), phospho-IRAK4 (Thr345/Ser346) (7652; CellSignaling Technology), Src (2102; Cell Signaling Technology),Phospho-Src (Tyr416) (2113; Cell Signaling Technology), BCL-2 (2870;Cell Signaling Technology) and Actin (4968; Cell Signaling Technology).

qRT-PCR

Total RNA was extracted using Trizol Reagent (Life Technologies) andreverse transcription was carried out using Superscript VILO (LifeTechnologies). Quantitative PCR was performed with Taqman Master Mix(Life Technologies) for human IRAK1 (Hs01018347_m1), TRAF6(Hs04185733_m1), GAPDH (Hs02758991_g1) on an Applied Biosystems StepOnePlus Real-Time PCR System.

shRNA-Mediated Knockdown of IRAK1

Lentiviruses were pseudotyped with VSV-G, produced by 293-FT cells, andconcentrated by ultracentrifugation at 20,000 rpm for 2 hours at 4° C.Cells at 1×105/mL were transduced with lentivirus at multiplicity ofinfection (MOI) of 0.5˜1 and in the presence of 8 μg/mL of polybrene(No. TR-1003-G; Millipore). At 48 hours post-transduction, GFP positivecells were isolated by fluorescence-activated cell sorting (FACS). ThepLKO.1 (OpenBiosystems) constructs were obtained from the Lentiviralcore at CCHMC and used to express shCTL (empty or scrambled control) andshIRAK1. Puromycin resistance gene was replaced by green fluorescentprotein (GFP). Two independent and validated pLKO.1-shIRAK1 constructswere obtained: TRCN0000000543 and TRCN0000000544. After validation,majority of experiments were performed with clone TRCN0000000544. Forinducible knockdown of IRAK1, we used the TRIPZ doxycycline(DOX)-inducible shRNA system (OpenBiosystems) expressing shIRAK1 cloneV2THS-132369.

Xenograftment of NOD/SCID-IL2Rγ(NSG) and NSG-hSCF/hGM-CSF/hIL3 (NSGS)MDSL cells (1×10⁶−5×10⁶) were injected into the tail veins of 8-week oldsublethally irradiated (250 Gy) NSG or NSG animals engineered to expresshuman SCF, GM-CSF, and IL3 (NSGS) as previously described (Wunderlich etal., 2010). Mice were monitored by performing complete blood counts(Drew Hemavet 950FS). Following red cell lysis, human MDSL cells wereidentified by hCD45-FITC staining. In vivo delivery of IRAK-Inh approachis adapted from previous reports (Yang et al., 2011): IRAK-Inh wasdiluted in DMSO (5 mM) and further dissolved in sterile phosphatebuffered saline (PBS; pH 7.2). Animals were injected i.p. with 2.12mg/kg IRAK-Inh 3× weekly.

Microarray Analysis

MDSL cells were transduced with lentivirus targeting human IRAK1 or anon-targeting control. At 2.5 days post-transduction, GFP positivepopulations were isolated by flow cytometry. Total RNA was extracted andpurified with Quick-RNA MiniPrep Kit (Zymogen). RNA quality was testedusing the Agilent Bioanalyzer 2100 (Hewelett Packard). Total RNA wasreverse transcribed and labeled, and hybridized onto the GeneChip HumanGene 1.0 ST Array (Affymetrix). Scanning was performed with theAffymetrix GeneChip Scanner 3000 7G and evaluated with the GenechipOperating Software (Affymetrix). Data mining was performed withGeneSpring software (Agilent). Gene set enrichment analysis wasperformed on a JAVA-based dataset supported by the Broad Institute(Subramanian et al., 2005). For evaluation of IRAK1 expression in MDS,previously published data sets were used (Pellagatti et al., 2006; Valket al., 2004). All microarray data files have been deposited in the GeneExpression Omnibus (accession number GSE46346).

Statistical Analysis

Results are depicted as the mean ±standard error of the mean.Statistical analyses were performed using Student's t-test. GraphPadPrism (v5, GraphPad) was used for statistical analysis.

Accession Numbers

The gene expression data from IRAK-Inh and IRAK1 RNAi can be found atthe GEO database (http.//www.ncbi.nlm.nih.gov/geo) using the accessionnumber GSE46346.

Primary Samples

This study was approved by the Cleveland Clinic (Cleveland, Ohio) and atFondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, University ofMilan (Milan, Italy). Informed consent was obtained according toprotocols approved by the review boards of participating institutions.The study conformed to the ethical standards set out in the Declarationof Helsinki. Diagnoses were reviewed at each of the participatingcenters and adapted, when required, to WHO 2008 criteria. For qRT-PCRanalysis, presentation bone marrow aspirates were collected from 20patients (CC1-CC20; Table 1). For functional studies, MDS mononuclearcells from bone marrow aspirates were obtained at diagnosis as part of amulticenter phase 2 trial based in Italy (MDS01-MDS08; Table 1). AMLpatient samples were acquired following informed consent and under thedirection of IRB approved protocols. Human CD34+ umbilical cord blood(UCB) and adult marrow-derived mononuclear cells were obtained fromCincinnati Children's Hospital.

Results

IRAK1 is Overexpressed and Activated in MDS

IRAK1 mRNA expression was evaluated in two gene expression studiescomparing normal and MDS CD34+ marrow cells (Hofmann et al., 2002;Pellagatti et al., 2010). Both studies revealed that IRAK1 transcript isoverexpressed by approximately 2-fold in ˜10-30% of MDS patients (FIG.1A; p=0.036 and p=0.05, respectively). An independent group of MDSpatients segregated according to high (top 50%) and low (bottom 50%)IRAK1 expression revealed that high IRAK1 expression correlates withreduced overall survival (p=0.035). IRAK1 protein expression wassimilarly overexpressed in marrow cells from 5 low/intermediate-risk MDSpatients, 3 AML patients, and in 6 MDS/AML cell lines (FIG. 1B-1E, Table1), suggesting that IRAK1 may be a relevant molecular target in MDS.

TABLE 1 Patient Characteristics ID Disease Risk OS MONTHS Gender AgeCytogenetics CC1 RCMD 0 8 Male 70 del(20)(q11.2)/46, idem,del(5)(q12q33) CC2 RCMD 0 51 Female 63 del(5)(q11.2q33), add(7)(q36),−9, der(14; 21)(q10; q10), add(17)(p13), 18, +21, +mar/47, idem, +2marCC3 5q-Syndrome 0 144 Female 57 del(5q) CC4 RCMD 1 24 Female 80del(5)(q15q33), del(7)(q22), −22 CC5 5q-Syndrome 0 21 Female 72del(5)(q15q33) CC6 RCMD-RS 0 5 Male 77 del(5)(q23q34), +6, +8, +9, +11,+19, +21 CC7 5q-Syndrome 0 21 Female 58 del(5)(q13) CC8 RAEB-1 0 5 Male58 del(5)(q22q35)[1]/45, idem, −7 CC9 RAEB-1 1 22 Female 67 del(3)(p14),−5, −7, +22 CC10 RCMD 1 17 Female 71 add(4)(q21), −5, add(8)(p23), −13,−16, add(17)(p11.2), −18 CC11 RAEB-2 1 11 Female 23 add(3)(q21), −5,t(7; 12)(q22; p13), −8, der(16)t(8; 16)(q13; q11.2) CC12 RAEB-2 1 2 Male75 add(3)(q11.2), del(5)(q13q33), +8, −15, −16 CC13 RAEB-1 0 26 Female78 del(5)(q15q33) CC14 RCMD 1 8 Male 79 del(1)(p21), del(3)(q21q26.2),inv(3)(q21q26.2), add(5)(q22), −6, add(7)(q22), −10, add(11)(q23), −12,−17 CC15 5q-Syndrome 0 46 Female 62 del(5q) CC16 RAEB-1 1 4 Male 58del(1), −3, −5, −7, +9, del(11)(p12) −18 CC17 RCMD 1 5 Male 67del(1)(p21), del(3)(q21q26.2), inv(3)(q21q26.2), add(5)(q22), −6,add(7)(q22), −10, add(11)(q23), −12, −17 CC18 5q-Syndrome 0 30 Female 75del(5q) CC19 RCMD-RS 0 10 Male 76 add(3)(p12), +add(3)(p12), −5,del(5)(q12q33), add(6)(p25), +8, +8, −17, −20, +22 CC20 RA 1 13 Female72 del(5)(q12q33), −7, del(7)(q22), del(7)(q22q34), −8, −12, −16, −16,−17, −18, +21 MDS01 5q-syndrome 0 na Female na del(5q) MDS02 5q-syndrome0 na Female na del(5q) MDS03 RAEB-1 0 na male 65 del(5q) MDS045q-syndrome 0 na female 83 del(5q) MDS05 RAEB-2 1 na male 62 na

IRAK1 is activated in response to lipopolysaccharide (LPS) orinterleukin-1β (IL-1β), and subsequently becomes phosphorylated (p) atthreonine-209 (pIRAK1T209) (FIG. 1F, 1G) (Kollewe et al., 2004). Todetermine the activation status of IRAK1 in MDS, we measured pIRAK1T209by immunoblotting marrow cells from 5 MDS patients. As shown in FIG. 1B,IRAK1 protein is not only overexpressed but also highly phosphorylatedat T209. To confirm these observations, we examined normal mononuclearcells (MNC), cord blood CD34+ cells, and a panel of 6 MDS/AML-derivedhuman cell lines. In accordance with MDS patients, IRAK1 isoverexpressed and hyperphosphorylated at T209 in all the MDS/AML celllines examined, but not in normal MNC or CD34+ cells (FIG. 1D, 1E). Incontrast, phosphorylated IRAK1 is observed to a lesser extent in primaryAML despite having overexpression of IRAK1 protein (FIG. 1C), suggestingthat activated IRAK1 is more pronounced and specific in MDS.Phosphorylation at Serine 376 (S376), a residue not implicated in IRAK1activation, was not phosphorylated in any of the cell lines (FIG. 1D).These findings indicate that IRAK1 is overexpressed and activated in MDSpatients.

The level of IRAK1 protein expression is significantly higher relativeto IRAK1 mRNA expression in MDS/AML cells, suggesting that IRAK1 isoverexpressed in part through a post-transcriptional effect (FIG. 1A,1B). Since IRAK1 is a validated target of miR-146a, a miRNA deleted andimplicated in the pathogenesis of MDS (Boldin et al., 2011;Starczynowski et al., 2010; Taganov et al., 2006), we evaluated whetherloss of miR-146a results in depression of IRAK1 protein in MDS cells. Wedesigned and overexpressed a retroviral miR-146a decoy in MDSL cells,which results in ˜80% downregulation of endogenous miR-146a. Knockdownof miR-146a in MDSL cells resulted in ˜3-fold increase in IRAK1 andTRAF6 protein (another validated miR-146a target). Furthermore, miR-146aexpression inversely correlated with total IRAK1 mRNA/protein andphosphorylated IRAK1 in MDS/AML cell lines and MDS patient cells.Although multiple mechanisms may contribute to increase IRAK1 expressionand/or activation, loss of miR-146a may represent a key event in MDSresulting in constitutively active IRAK1. Notwithstanding,overexpression and activation of IRAK1 is a common feature in MDS.

IRAK-Inh Blocks TRAF6 and NF-κB Activation.

A small molecule inhibitor of IRAK1 (IRAK1/4-Inh or IRAK-Inh), whichselectively inhibits its kinase activity in a low micromolar range(IC50=0.75 μM), has been initially developed for autoimmune disease(FIG. 2A) (Powers et al., 2006; Wang et al., 2009). To determine whetherIRAK-Inh can effectively inhibit active IRAK1 in MDS/AML cell lines andpatient cells, we treated cells with an escalating dose of IRAK-Inh(0-10 μM) for 24 hrs. pIRAK1T209 was reduced in a dose-dependent mannerin MDS/AML cell lines (FIG. 2B). At 10 μM IRAK-Inh, phosphorylated IRAK1was reduced by ˜70% in cell lines and patient marrow cells (FIG. 2B-2D).Examination of kinases with structural homology to IRAK1 revealed thatonly IRAK1 is a target of IRAK-Inh in malignant myeloid cells.Lentiviral-mediated IRAK1 or TRAF6 overexpression resulted in increasedpIRAK1T209 (without exogenous stimulation), which is also inhibited byIRAK-Inh by ˜50%. Upon phosphorylation, IRAK1 simultaneously undergoesTRAF6-mediated K63-linked ubiquitination (FIG. 2A), which is anotherindicator of its active state (Conze et al., 2008). In the presence ofIRAK-Inh, immunoprecipitated (IP) IRAK1 exhibits reduced phosphorylationand K63-linked ubiquitination (FIG. 2E).

TRAF6 forms a signaling complex with pIRAK1, resulting in IKK complexactivation and subsequent NF-κβ (RelA/p65) nuclear DNA-binding (FIG.2A). As expected, TRAF6 overexpression induces phosphorylation ofIKKα/IKKβ, the two catalytic proteins within the IKK complex (FIG. 2F).In the presence of IRAK-Inh (10 μM for 24 hrs), vector- andTRAF6-expressing cells exhibit reduced pIKKα/IKKβ (FIG. 2F), but notrelevant MAP kinases (p38 or ERK). Inhibition of p100/p52 processing,which is a measure of non-canonical NF-κβ activation and is independentof TRAF6 (FIG. 2A), was also completely blocked by the IRAK-Inh (FIG.2A, 2F). In addition, DNA bound and active RelA/p65 was decreased by˜50% in MDS/AML cell lines by IRAK-Inh, indicating that IRAK-Inheffectively blocks IRAK1-mediated activation of NF-κB in MDS/AML cells(FIG. 2G). Lastly, TRAF6 undergoes K63-autoubiquitination, which is anecessary step prior to NF-κB activation (FIG. 2A). Treatment withIRAK-Inh also coincides with reduced polyubiquitinated TRAF6 (FIG. 2H).Taken together, IRAK-Inh effectively blocks IRAK1 function as is evidentby reduced levels of phosphorylated and K63-ubiqutinated IRAK1, reducedautoubiquitination of TRAF6, and impaired NF-κβ nuclear DNA binding(FIG. 2A).

Cytostatic Effect of IRAK-Inh on MDS Progenitor Function and CellGrowth.

MDS/AML cell lines with hyperphosphorylated IRAK1 (FIG. 1D) wereevaluated for sensitivity to IRAK-Inh. MDS/AML cell lines exposed toincreasing concentration of IRAK-Inh were cultured for up to 6 days invitro (FIG. 3A). A significant, dose-dependent impairment of MDSL, TF1,and THP1 cell proliferation was observed in the presence of IRAK-Inh(FIG. 3A). In contrast, the proliferation of normal cord blood-derivedCD34⁺ cells, which do not exhibit activation of IRAK1, was not affectedwith even high doses of IRAK-Inh (FIG. 3A). Despite havinghyperphosphorylated IRAK1 (FIG. 1C, 1D) and exhibiting reduced pIRAK1after IRAK-Inh treatment (FIG. 2D), the viability of HL60 cells andprimary AML marrow cells was only modestly reduced with IRAK-Inh (FIG.3A). In support of this observation, the inhibitory effect of IRAK-Inhon the cell lines correlated with the level of phosphorylated IRAK1(R2=0.36; FIG. 3B). In parallel, cell viability was examined after 48hrs of treatment with the IRAK-Inh. All cell lines exhibited a modestincrease in apoptosis (FIG. 3C). To investigate whether IRAK-Inh affectscell cycle progression, MDSL cells were treated with 10 μM IRAK-Inh forup to 6 days and evaluated by BrdU/7AAD staining (FIG. 3D). Consistentwith reduced proliferation (FIG. 3A), MDSL cells treated with IRAK-Inhhave altered cell cycle kinetics: there are fewer viable MDSL cells in Sphase (4.3%±0.4 versus 14.2%±0.2; p=0.0002) and increased proportion inG0/G1 (62.7%±1.1 versus 54.5%±0.7; p=0.008) (FIG. 3D). In addition, theproportion of sub-G0 cells was significantly increased inIRAK-Inh-treated cells (5.4%±0.3 versus 1.8%±0.2; p=0.003).

The effect of IRAK-Inh on leukemic progenitor function was evaluated inmethylcellulose containing IRAK-Inh. Normal CD34⁺ formed equivalentnumber of colonies with moderate skewing of erythroid andgranulocytic/macrocytic progenitors (p<0.05) when treated with IRAK-Inh.In stark contrast, all MDS/AML cells formed significantly fewer colonieswhen treated with IRAK-Inh (FIG. 3E). As an alternative approach,MDS/AML cells were pre-treated with IRAK-Inh for 24 hours and thenplated in methylcellulose. After transient exposure (24 hr) to IRAK-Inh(10 μM), MDS/AML cells, but not normal CD34⁺, formed significantly fewercolonies, suggesting that IRAK-Inh suppresses the function of thedisease-propagating cell. Increased apoptosis, impaired cell cycleprogression, and reduced progenitor function after IRAK-Inh treatment isnot a consequence of induced differentiation, as IRAK Inh-treatedMDS/AML cells did not undergo noticeable myeloid differentiation invitro. To determine whether the effects of IRAK-Inh can be rescued byactivating the innate immune pathway downstream of IRAK1 (FIG. 2A), weoverexpressed TRAF6 and then measured the cellular toxicity of IRAK-Inh.Although IRAK-Inh partially suppresses NF-κβ in TRAF6-overexpressingcells (FIG. 2F), forced expression of TRAF6 in MDSL cells can overcomethe inhibitory effects of IRAK-Inh and restore cell viability andprogenitor function in IRAK Inh-treated cells. Collectively, theseresults suggest that IRAK-Inh is effective in selectively inhibiting theviability and function of MDS progenitor cells while sparing normalCD34⁺ cells by directly targeting the innate immune pathway.

We evaluated marrow cells from 7 MDS and 3 AML patient samples withvarious cytogenetic features to determine whether IRAK1 inhibition isalso effective in primary patient cells (Table S1). MDS marrow cellstreated with IRAK-Inh for 48 hrs failed to expand (FIG. 3F) andexhibited increased levels of apoptotic cells, except for MDS-07 (FIG.3G). In contrast, vehicle control-treated MDS cells expanded nearly2-fold during this time and had significantly fewer apoptotic cells(FIG. 3F, 3G). AML marrow cells treated with IRAK-Inh continued to growsimilar to control-treated cells. Since MDS and AML stem/progenitorcells are clonal and the disease-propagating cells form colonies inmethylcellulose, we also evaluated the effects of IRAK-Inh on colonyformation. Whether continuously treated (FIG. 3H) or briefly exposed toIRAK-Inh, MDS marrow cells formed significantly fewer colonies in thepresence of IRAK-Inh. In contrast, IRAK-Inh did not affect AML orcontrol CD34+ cell progenitor function (FIG. 2H). These findingsindicate that IRAK-Inh selectively inhibits growth and progenitorfunction of primary MDS marrow cells, and that IRAK-Inh sensitivity is ageneral feature of MDS, independent of existing genetic features.

IRAK-Inh Ameliorates Disease in a Human Xenograft Model Using anMDS-derived Cell Line.

Primary MDS patient samples remain difficult to engraft intoimmunodeficient mice, typically with less than 5% marrow engraftment andno evidence of disease (Martin et al., 2011; Park et al., 2011). Tocircumvent this limitation, we developed a xenograft model using an MDSpatient-derived cell line (MDSL), which has retained phenotypic andcellular characteristics of MDS (Matsuoka et al., 2010; Tohyama et al.,1995). Consistent with non-transforming MDS subtypes, the MDSL cell linedoes not form overt leukemia in NSGS or NSG mice. Instead, MDSL cellsengraft into the marrow and gradually expand over time within themarrow, spleen, and peripheral blood as a non-blast/incompletelydifferentiating myeloid population. Xenografted mice develop progressiveanemia, thrombocytopenia, and extramedullary hematopoiesis andeventually succumb to disease. Disease progression and MDSL expansion inthe marrow is accompanied by depletion of normal mouse hematopoieticcells and a hypocellular marrow.

To determine whether IRAK-Inh can delay human MDS-like disease in vivo,MDSL were pre-treated with IRAK-Inh (10 μM) for 24 hrs in vitro andsubsequently injected intravenously (i.v.) into NSG (5×10⁶ cells) andNSGS (1×10⁶ cells) recipient mice (FIG. 4A). This approach permitted usto evaluate the cell autonomous effect of IRAK-Inh on MDS cell viabilityand engraftment without altering endogenous IRAK1 function in the mouse.As shown in FIG. 4B, pretreatment with IRAK-Inh significantly delayedthe MDS-like disease in NSG mice (median survival=80 days vs 68 days;p=0.0065) and in NSGS mice (median survival=38 days vs 29 days;p=0.028). NSGS mice transplanted with IRAK-Inh-treated MDSL cells alsohad significantly improved red blood cell numbers (p=0.0085), hemoglobin(p=0.012), hematocrit (p=0.015) and platelets (p=0.02) (FIG. 4C). Inaddition, the human MDSL graft was reduced from 8% to 2% (FIG. 4D).Morphological assessment confirmed normal RBC and reduced MDS grafts inthe peripheral blood and marrow of mice transplanted withIRAK-Inh-treated MDSL cells (FIG. 4E). To demonstrate that the IRAK-Inhcan also ameliorate disease after cells have engrafted, MDSL wereinjected i.v. into NSG mice, followed by intraperitoneal (i.p.)injection of IRAK-Inh (FIG. 4A). Mice receiving IRAK-Inh maintained HCTand Hb levels while control mice exhibited a progressive anemia (FIG.4F). Within ˜7 days of receiving IRAK-Inh, mice had reduced human graftin the peripheral blood (FIG. 4G, 4H). These findings indicate thatIRAK-Inh targets the disease-propagating cell and provides a significantsurvival benefit in a xenograft model of human MDS.

Knockdown of IRAK1 Protein Induces Apoptosis and ImpairedClonal-progenitor Function.

To validate the effects and specificity of the IRAK-Inh, lentiviralvectors encoding independent short hairpin RNAs (shRNA) targeting IRAK1were transduced into MDS/AML cell lines and MDS patient marrow cells. Weconfirmed targeting of IRAK1 by immunoblotting and quantitative RT-PCR(qRT-PCR) (FIG. 5A). Although IRAK-Inh induced only a modest apoptoticaffect (FIG. 3B), all MDS/AML cell lines with depletion of IRAK1correlated with a significant increase in apoptosis (FIG. 5B). Knockdownof IRAK1 in CD34⁺ cells did not induce apoptosis, indicating that IRAK1is dispensable for normal CD34⁺ viability (FIG. 5B). MDS/AML cell linesand MDS patient samples with knockdown of IRAK1 also exhibited asignificant decrease in progenitor colonies in methylcellulose (FIG. 5C,5D). Normal CD34⁺ were not sensitive to loss of IRAK1 and formedcolonies at the same level to control-transduced CD34⁺ cells (FIG. 5C).To examine the effects of IRAK1 depletion in vivo, MDSL cells weretransduced with a doxycycline (DOX)-inducible shIRAK1 (pTRIPZ).Increasing amounts of DOX in vitro demonstrated a dose-dependentdeletion of IRAK1 mRNA and protein, as well as reduced cell viability(FIG. 5E). The effects of IRAK1 knockdown were rescued by expressing ahairpin-resistant IRAK1 cDNA. Transduced MDSL cells were injected intoNSG mice, and six days post-transplant, mice were given chow with orwithout DOX. Expression of the shRNA is observed in the marrow, spleen,and blood of DOX-treated mice, but not expressed in control mice.Without IRAK1 knockdown (minus DOX or non-transduced parental MDSLcells), mice developed the MDS-like disease (FIG. 5F-5I). In contrast,IRAK1 knockdown (plus DOX) reduced engraftment of MDS cells (PB: ˜15%versus 5% [FIG. 5F]; BM: ˜40% vs 10% [FIG. 5G]) and spleen size (FIG.5H), and significantly delayed mortality in mice (p=0.001; FIG. 5I).Collectively these data suggest that genetic depletion of IRAK1 resultsin reduced viability and growth of MDS/AML progenitor cells in vitro andin vivo. In addition, a more profound effect on MDS/AML survival andprogenitor function was observed following depletion of IRAK1 ascompared to the IRAK-Inh.

Expression Profiling Following Deletion or Inhibition of IRAK1 RevealsOverlapping Gene Signatures and a Compensatory Increase in BCL2

We performed a gene expression analysis to (1) gain insight into themolecular consequences of inhibiting IRAK1 in MDS cells, and (2) definean underlying mechanism for the discrepancy between IRAK-Inh and shIRAK1apoptotic threshold in MDS/AML cells. MDSL cells were either transducedwith shIRAK1 (or control) or treated with IRAK-Inh (or DMSO) for 48hours, followed by RNA collection for a microarray analysis (FIG. 6A).At this time point, we observe >50% knockdown of IRAK1 mRNA by the shRNAand minimal effect on cell viability. Inhibition of IRAK1 by eitherapproach in MDSL resulted in ˜180 differentially expressed genes (FIG.6A). We searched for previously defined expression signatures thatoverlap genes regulated by both IRAK-Inh and shIRAK1 by using gene setenrichment analysis (GSEA) (Subramanian et al., 2005) (FIG. 6A). Of thetop 10 significant GSEA sets in each group, 3 gene sets were identicalbetween IRAK-Inh and shIRAK1 MDSL cells (FIG. 6B), indicating that IRAK1is effectively targeted by both approaches and that inhibition by eitherapproach induced a similar transcriptional response. Of the top GSEAsets, survival (IL6_Starve_Up) and cell cycle/proliferation (Cell_Cycle,and MM_CD138_PR_vs_REST) were the most significant in both IRAK-Inh andshIRAK1 experimental groups (FIG. 6B). To better understand the cellularand molecular consequences following IRAK1 inhibition/deletion, weexamined the Gene Ontology categories using ToppGene (Chen et al.,2009). Knockdown of IRAK1 resulted in down regulation of genes involvedin chromatic assembly, DNA binding, and RNA metabolism (FIG. 6C).IRAK-Inh treatment resulted in downregulation of genes involved in cellmigration and adhesion, inflammatory response, and cytokine-mediatedsignaling (FIG. 6C).

Despite the overlap in gene sets, we examined genes that could explainthe discrepancy between IRAK-Inh and shIRAK1 in inducing apoptosis ofMDS/AML cells. Although IRAK-Inh upregulated pro-apoptotic genes (e.g.,BIM, CASP10, RIPK1), downregulation of anti-apoptotic Bcl2-family geneswas not evident. As an example, BCL2 mRNA and protein was notdownregulated in IRAK-Inh-treated cells; surprisingly, BCL2 expressionwas increased in several of the cell lines examined, an effect notobserved in shIRAK1-expressing cells (FIG. 6D-6F). This observationprompted us to speculate that a subset of MDS/AML progenitors escapeIRAK-Inh induced apoptosis because of a compensatory upregulation orinefficient downregulation of BCL2.

Combined Inhibition of IRAK1 and BCL2 Cooperates to Target MDSClonal-progenitor Function.

We examined the survival dependence on BCL2 function in IRAK-Inh-treatedcells by utilizing a BH3-mimetic (ABT-263, Abbott Laboratories).Administration of IRAK-Inh (10 μM) or ABT-263 (0.1 μM) alone had modesteffects in inhibiting MDS/AML cell line growth and survival (FIG. 7A,7B). Strikingly, co-treatment of the MDS/AML cells with IRAK-Inh andABT-263 significantly and synergistically inhibited cell growth andsurvival (FIG. 7A, 7B). In particular, HL60 cells, which were refractoryto the inhibitory effects of IRAK-Inh or ABT-263 alone, are extremelysensitive to the combined treatment with ABT-263 (FIG. 7A, 7B). Inaddition, MDS/AML cell lines and MDS patient progenitor colonies weresignificantly impaired with the combination treatment of IRAK-Inh andABT-263 (FIG. 7C, 7D). For AML patient cells that are not sensitive toIRAK-Inh treatment alone, co-treatment with ABT-263 also resulted inreduced leukemic progenitor function (FIG. 7E, 7F). The viability andhematopoietic progenitor function in methylcellulose of normal CD34+cells was not affected by the individual or combination treatment ofIRAK-Inh and ABT-263.

Moreover, we investigated whether combined IRAK1 and BCL2 inhibitioncould delay human MDS-like disease in vivo. MDSL cells were treated withIRAK-Inh (10 μM) and/or ABT-263 (0.1 μM) for 48 hrs in vitro andsubsequently injected i.v. into NSGS mice (FIG. 4A). Treatment withIRAK-Inh or ABT-263 alone significantly delayed the MDS-like disease inNSG mice with a median survival of ˜35 days (versus 28 days with DMSOtreatment) (FIG. 7G). Mice receiving MDSL co-treated with IRAK-Inh andABT-263 exhibited a significantly enhanced survival (43 days) ascompared to individual drugs or DMSO (FIG. 7G). Not only was survivalextended, but mice transplanted with cells pre-treated with the drugcombination had improved red blood cell, hemoglobin, and platelet counts(not shown). In conclusion, inhibition of IRAK1 function with asmall-molecular inhibitor may represent a treatment to inhibit MDS clonefunction and viability, while co-treatment with ABT-263 results inenhanced cytotoxicity.

Discussion

IRAK1 is a serine/threonine kinase that mediates signals from TLR/IL1Rto NF-KB under normal conditions (Flannery and Bowie, 2011). Followingreceptor activation, MyD88 recruits IRAK4 and IRAK1, resulting in IRAK1hyperphosphorylation. IRAK1 phosphorylation at Thr-209, which ismediated by upstream signals or through autophosphorylation, is a keypost translational modification and a hallmark of its activation(Kollewe et al., 2004). Once phosphorylated, IRAK1 binds TRAF6 andundergoes K63-linked ubiquitination (Conze et al., 2008). Thisinteraction between IRAK1 and TRAF6 initiates a signaling cascaderesulting in NF-κβ nuclear translocation (Conze et al., 2008). Smallmolecule inhibitors targeting IRAK1 have been originally developed forautoimmune and inflammatory diseases (Durand-Reville et al., 2008; Wanget al., 2009). Given that the TRAF6/IRAK1 signaling complex remains inan activated state in MDS (Fang et al., 2012; Starczynowski and Karsan,2010; Starczynowski et al., 2010), it is not surprising that inhibitingthis complex may have a therapeutic benefit in MDS, and represent aviable approach to inhibit NF-κβ preferentially in malignant clones withactivated IRAK1. Notably, most NF-κβ inhibitors to date have beendisappointing for the treatment of myeloid malignancies due to toxicity(Breccia and Alimena, 2010).

Although IRAK1 mRNA is overexpressed in a subset of MDS patients, thelevel of expression rarely exceeds 2-fold. However, deletion and reducedexpression of miR-146a is a common event in MDS as it resides within thedeleted region on chr 5q and its expression is reduced in a large subsetof normal karyotype MDS (Starczynowski et al., 2010; Starczynowski etal., 2011b). TRAF6 and IRAK1 are two targets of miR-146a, and germlineknockout of miR-146a results in derepression of TRAF6 and IRAK1 protein(Boldin et al., 2011; Zhao et al., 2011). We confirmed a similar effectin MDS cells. This suggests that IRAK1 is transcriptionally andtranslationally upregulated in MDS patients making it a relevantmolecular target. IRAK1 mRNA/protein is also overexpressed in subsets ofAML patients (Camos et al., 2006), supporting our observations thattargeting IRAK1 may extend to high-risk MDS and AML with active IRAK1.

Phosphorylation and activation of IRAK1 can also occur bygain-of-function mutations or aberrant expression of upstream signalingmolecules. For example, human lymphomas with oncogenically active MyD88mutations have constitutive IRAK1 phosphorylation and NF-κβ activation,and are sensitive to IRAK inhibitors (Ngo et al., 2010). In Fanconianemia, IRAK1 exists in a hyperphosphorylated (higher-molecular weight)state, potentially as a consequence of aberrant TLR8 signaling(Vanderwerf et al., 2009). Of note, mutations in MyD88 or TLR8 have notbeen reported in MDS or AML, suggesting that alternate molecularalterations activate IRAK1 in MDS/AML. However, mutations of TLR2 arereported in ˜10% of MDS patients (Wei et al., 2012), and a recentfinding identified overexpression of interleukin-1 receptor accessoryprotein (IL1RAP) in HSC from MDS and AML patients (Barreyro et al.,2012). Consistent with the hypothesis that hyperphosphorylation of IRAK1in MDS may be due to aberrant activation downstream of the TLR/IL1R, aretrospective analysis revealed that chronic immune stimulation acts asa trigger and increases the risk for MDS and AML development(Kristinsson et al., 2011). Collectively, these observations suggestthat, in addition to downregulation of miR-146a, multiple othermolecular alterations can converge on and activate IRAK1 in MDS.

According to our integrative gene expression analysis, inhibition ofIRAK1 revealed that IRAK1 regulates genes involved in survival, cellcycle/proliferation, chromatic assembly/DNA binding, RNA metabolism,cell migration/adhesion, and inflammation (FIG. 6C). These genesignatures are consistent with our observation that inhibiting IRAK1 inprimary MDS marrow or cell lines results in delayed proliferation,reduced survival, and impaired progenitor function. IRAK-Inh wasinefficient at downregulating pro-survival BCL2 genes, and in some celllines, resulted in a compensatory increase in BCL2 expression, which isa common cellular response to cytostatic and cytotoxic therapies(Thomadaki and Scorilas, 2008). To overcome this compensatory effect, wecombined a BCL2 inhibitor (ABT-263) with IRAK-Inh, which resulted inpotent collaborative cytotoxic effects in MDS and AML cells by inducingrapid and profound apoptosis. Notably, this effect was observed even inMDS/AML cells that did not exhibit a compensatory increase in BCL2expression in response to IRAK-Inh. That IRAK-Inh is effective atsuppressing MDS cells, but not AML, can be explained by: (1) anincreased apoptotic threshold in high-risk MDS and AML due to higherexpression of prosurvival BCL2 family members, thus necessitatingco-treatment with a BCL2 inhibitor; (2) the level of activated IRAK1 inAML is lower as compared to MDS, suggesting differences in the molecularcircuitry of IRAK1 activation; and/or (3) only select subtypes of AMLare sensitive to IRAK-Inh while a larger proportion of MDS aresensitive.

The complexity and heterogeneity of MDS, and the lack of human xenograftmodels remain as obstacles to identifying and evaluating novel moleculartargets for this disease. In addition, primary MDS cells do notefficiently engraft into immunodeficient mice (Martin et al., 2011; Parket al., 2011). To overcome this limitation, we generated a human modelusing an MDS-derived patient cell line (MDSL). Consistent withphenotypic and cellular characteristics of MDS (Matsuoka et al., 2010;Tohyama et al., 1995), MDSL engraftment into NSG or NSGS immunodeficientmice results in a fatal and progressive anemia, thrombocytopenia,hypocellular marrow, and extramedullary hematopoiesis. Treatment of MDSLcells with IRAK-Inh or in vivo delivery of the inhibitor reduced thenumber of MDSL cells and delayed disease. This xenograft modelrepresents an alternative to examine the mechanisms of low-risk MDSdisease and a tool for preclinical studies using an MDS-derived patientcell line.

In summary, this work implicates IRAK1 as a drugable target for MDS.Inhibition with IRAK-Inh induces combined apoptosis and a cell cycleblock, while inhibition with ABT-263 results in collaborativecytotoxicity in MDS cells Inhibitors targeting IRAK1 reveal an avenuefor suppressing altered TLR/IL1R/TRAF6/NF-κB pathway and eliminating theMDS clone.

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All percentages and ratios are calculated by weight unless otherwiseindicated.

All percentages and ratios are calculated based on the total compositionunless otherwise indicated.

It should be understood that every maximum numerical limitation giventhroughout this specification includes every lower numerical limitation,as if such lower numerical limitations were expressly written herein.Every minimum numerical limitation given throughout this specificationwill include every higher numerical limitation, as if such highernumerical limitations were expressly written herein. Every numericalrange given throughout this specification will include every narrowernumerical range that falls within such broader numerical range, as ifsuch narrower numerical ranges were all expressly written herein.

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “20 mm” is intended to mean“about 20 mm.”

Every document cited herein, including any cross referenced or relatedpatent or application, is hereby incorporated herein by reference in itsentirety unless expressly excluded or otherwise limited. The citation ofany document is not an admission that it is prior art with respect toany invention disclosed or claimed herein or that it alone, or in anycombination with any other reference or references, teaches, suggests ordiscloses any such invention. Further, to the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed:
 1. A method of treating myelodysplastic syndrome (MDS)characterized by increased NFκB activity in an individual comprising thestep of administering to said individual a composition comprising anIRAK1/4 inhibitor.
 2. The method of claim 1 wherein said IRAK1/4inhibitor is selected from N-acyl-2-aminobenzimidazoles,imidazo[1,2-a]pyridino-pyrimidine, imidazo[1,2-a]pyridino-pyridine,benzimidazolo- pyridine,N-(2-morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole,

or combinations thereof.
 3. The method of claim 1 wherein said IRAK1/4inhibitor comprises an RNAi sufficient to inhibit IRAK1 expression. 4.The method of claim 1, further comprising the step of administering tosaid individual an apoptotic modulator.
 5. The method of claim 1,further comprising the step of administering to said individual anapoptotic modulator, wherein said apoptotic modulator comprises a BCL2inhibitor.
 6. The method of claim 1, comprising the step ofadministering to said individual an apoptotic modulator, wherein saidapoptotic modulator comprises a BCL2 inhibitor selected from

combinations thereof.
 7. The method of claim 1 wherein saidmyelodysplastic syndrome is selected from Fanconi Anemia, refractoryanemia, refractory neutropenia, refractory thrombocytopenia, refractoryanemia with ring sideroblasts (RARS), refractory cytopenia withmultilineage dysplasia (RCMD), refractory anemia with excess blasts Iand II (RAEB), 5q- syndrome, myelodysplasia unclassifiable, refractorycytopenia of childhood, or a combination thereof.
 8. The method of claim1 wherein said administering step is selected from orally, rectally,nasally, topically, parenterally, subcutaneously, intramuscularly,intravenously, transdermally, or a combination thereof.
 9. The method ofclaim 1 wherein said administration decreases the incidence of marrowfailure, immune dysfunction, transformation to overt leukemia, orcombinations thereof in said individual, as compared to an individualnot receiving said composition.
 10. The method of claim 1 wherein saidmethod decreases a marker of viability of MDS cells.
 11. The method ofclaim 1, wherein said treatment decreases a marker of viability of MDScells.
 12. The method of claim 1, wherein said treatment decreases amarker of viability of MDS and/or AML cells, wherein marker is selectedfrom survival over time, proliferation, growth, migration, formation ofcolonies, chromatic assembly, DNA binding, RNA metabolism, cellmigration, cell adhesion, inflammation, or a combination thereof.
 13. Amethod of treating myelodysplastic syndrome (MDS) or acute myeloidleukemia (AML), wherein said MDS or AML is characterized by increasedNFκB activity, in an individual comprising the step of administering tosaid individual a) an IRAK1/4 inhibitor; and b) an agent selected froman apoptotic agent, an immune modulating agent, an epigenetic modifyingagent, and combinations thereof.
 14. The method of claim 13 wherein saidIRAK1/4 inhibitor is selected from N-acyl-2-aminobenzimidazoles,imidazo[1,2-a]pyridino-pyrimidine, imidazo[1,2-a]pyridino-pyridine,benzimidazolo- pyridine,N-(2-morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole,

or combinations thereof.
 15. The method of claim 13 wherein said IRAK1inhibitor comprises an RNAi sufficient to inhibit IRAK1 expression. 16.The method of claim 13 wherein said administration step includesadministration of an apoptotic modulator.
 17. The method of claim 13wherein said administration step includes administration of an apoptoticmodulator comprising a BCL2 inhibitor.
 18. The method of claim 13wherein said administration step includes administration of an apoptoticmodulator selected from

combinations thereof.
 19. The method of claim 13 wherein saidadministration step includes administration of an immune modulator. 20.The method of claim 13 wherein said administration step includesadministration of an immune modulator, wherein said immune modulatorcomprises lenalidomide.